1. Field
This invention is concerned generally with identifying protein-ligand interactions, and specifically with peptide ligands which bind rFVIII and which may be used in a method for the affinity purification of rFVIII.
2. Prior Art
Human Factor VIII, (antihemophilic factor) is a human plasma protein consisting of 2 polypeptides (light chain molecular weight of 80,000 and heavy chain molecular weight variable from 90,000 to 220,000). It is an essential cofactor in the coagulation pathway; required for the conversion of Factor X into its active form (Factor Xa). Factor VIII circulates in plasma as a non-covalent complex with von Willebrand Factor. Blood concentrations of Factor VIII below 20% of normal cause a bleeding disorder designated hemophilia A. Factor VIII blood levels less than 1% of normal result in a severe bleeding disorder, with spontaneous joint bleeding being the most common symptom. Recombinant DNA technology has allowed construction of plasmids that direct the expression of fusion products of Factor VIII protein in transfected mammalian cells. Factor VIII can be isolated from either a plasma derived source (cryoprecipitate) or from a genetically engineered recombinant source. The term Factor VIII is not meant to be a limitation but refers to a functional protein for treating bleeding disorders.
Several methods have been described for purification of Factor VIII from plasma sources (1,2,3). Several purification schemes utilize antibody affinity columns (4,5). To date, the most successful purifications of Factor VIII from plasma or from recombinant sources has been accomplished by using murine monoclonal antibodies specific to either Factor VIII or von Willibrand Factor.
Although monoclonal antibodies have been used successfully to obtain a relatively pure Factor VIII preparation, monoclonal antibodies can be present in the Factor VIII effluent because of leaching from the support matrix. This raises the possibility of antigenicity when the final preparation is introduced into animal systems, since murine monoclonal antibodies have been shown to be antigenic. A second disadvantage of the use of monoclonal antibodies is the requirement of cell culture facilities for producing the antibodies and the concomitant cost of purification and attachment onto a support matrix. Finally, the stability of the antibody binding site may not be amenable to the rigorous conditions necessary to sanitize the column.
Affinity chromatography is one of the most efficient techniques for purifying a protein from a complex mixture. With potential advantages including high stability, efficiency, selectivity, and low price, peptides have been investigated as affinity ligands in the pharmaceutical industry. A recent approach for identifying such ligands is to screen peptides from combinatorial peptide libraries (6,7,8,9).
Using the `divide-couple-recombine` approach (10,11,12), millions of unique peptides of a defined length may be synthesized on resin beads. Each bead contains a unique peptide sequence. These library beads and their corresponding peptide sequences are then exposed to a target protein. Among these millions of peptide sequences, the target protein may bind to several unique bead-sequences. Those beads and their corresponding sequences must be detected, isolated, and identified. Several detection systems, including calorimetric two-step methods (7,12,13) as well as direct fluorescence detection methods (14,15,16) have been used.
Peptides disclosed in the U.S. patent application Ser. No. 08/595,718, incorporated herein by reference, also were found to bind Factor VIII. See also Necina et al. (23)